title: Structure-function studies of Escherichia coli RnlA reveal a novel toxin structure involved in bacteriophage resistance.
abstract: Escherichia coli RnlA-RnlB is a newly identified toxin-antitoxin (TA) system that plays a role in bacteriophage resistance. RnlA functions as a toxin with mRNA endoribonuclease activity and the cognate antitoxin RnlB inhibits RnlA toxicity in E. coli cells. Interestingly, T4 phage encodes the antitoxin Dmd, which acts against RnlA to promote its own propagation, suggesting that RnlA-Dmd represents a novel TA system. Here, we have determined the crystal structure of RnlA refined to 2.10  (Dmd-binding domain), which is an organization not previously observed among known toxin structures. Small-angle X-ray scattering (SAXS) analysis revealed that RnlA forms a dimer in solution via interactions between the DBDs from both monomers. The in vitro and in vivo functional studies showed that among the three domains, only the DBD is responsible for recognition and inhibition by Dmd and subcellular location of RnlA. In particular, the helix located at the C-terminus of DBD plays a vital role in binding Dmd. Our comprehensive studies reveal the key region responsible for RnlA toxicity and provide novel insights into its structure-function relationship.
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, 230027, China; Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wei
authors.forename: Zeng-Qiang
authors.initials: ZQ
authors.lastname: Gao
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Otsuka
authors.forename: Kenta
authors.initials: K
authors.lastname: Naka
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Yonesaki
authors.forename: Heng
authors.initials: H
authors.lastname: Zhang
authors.forename: Yu-Hui
authors.initials: YH
authors.lastname: Dong
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D048168:Escherichia coli K12 / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; C453723:Dmd protein, Enterobacteria phage T4; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C525943:RnlA protein, E coli; D014764:Viral Proteins; C557568:rnlB protein, E coli